This invention relates to a method for the immobilization of an enzyme by use of a water-soluble photo-crosslinking resin of high sensitivity. Because they do little to cause environmental pollution, water-soluble photosensitive resins are used as photoresists and photo-milling agents. In recent years, the potentiality of their adoption for the immobilization of bio-active materials such as enzymes has come to attract increasing attention.
The methods heretofore proposed to the art for the purpose of immobilization of enzymes are broadly divided into three types; the type involving the binding of enzymes with carriers, the type involving the crosslinking between enzyme molecules and the type involving the entrapment of enzymes.
Of these methods, the carrier-binding methods accomplish the immobilization of a given enzyme by causing the enzyme to be chemically or physically bound to a suitable carrier. By the manner of binding involved, the methods of this type are sub-divided into a covalent binding method, an ionic binding method, a physical absorption method, etc. These methods, however, have a common disadvantage that in most cases, the steric structure of the enzyme under treatment particularly near the center of activity and the state of the enzyme's electric charge are affected and, consequently, the enzyme's activity is degraded. In immobilizing a particular enzyme, therefore, it has been necessary to examine all the methods and select the optimum method by the principle of trial and error.
The crosslinking methods generally accomplish the required immobilization of a given enzyme by causing the individual enzyme molecules to be covalently bound with one another and, therefore, have an inevitable consequence that the immobilized enzyme exhibits considerably degraded activity compared with the enzyme in its unimmobilized state.
The methods of the type involving the entrapment of a given enzyme are sub-divided into a method which entraps the enzyme in a high-molecular matrix and a method which seals the enzyme in micro-capsules. These methods are capable of immobilizing given enzymes without impairing the molecular structures of the enzymes and, therefore, are attracting attention as highly promising techniques. Particularly the method which entraps the enzyme in a high-molecular matrix is especially noteworth because it can confine the enzyme molecules within the molecules of the high-molecular matrix in much the same way as animals are locked up in cages and, thus, accomplish the immobilization of the enzyme without spoiling the function inherent in the enzyme or impeding a given substrate from obtaining access to the entrapped enzyme.
The methods of the type involving the entrapment of enzymes in high-molecular matrices which have heretofore been proposed to the art include a method which causes a water-soluble high-molecular compound to be thermally crosslinked, a method which causes a water-soluble high-molecular compound to be crosslinked by exposure to radiant rays, a method which causes a vinyl monomer or oligomer to be radically polymerized (G. P. Hicks and S. J. Updike; Anal. Chem., 38, 726, 1966), a method which causes a similar vinyl monomer or oligomer to be photopolymerized (S. Fukui et al; FEBS LETTERS, Vol. 66, No. 2, p. 179 July, 1976) and a method which causes a water-soluble high-molecular compound to be photo-crosslinked through the agency of bis-azide (U.S. Pat. No. 4,160,698), for example.
Of these conventional methods, those methods which accomplish the crosslinking of high-molecular compounds through the agency of thermal or radiant energy have a possibility of causing the deactivation due to the chemical change in the enzyme molecules under treatment and the two methods involving the exposure of matrix compounds to light inevitably use for the purpose of crosslinking reaction those free radicals and nitrene which are highly reactive and therefore devoid of selectivity and, as the result, fall to have enzymes entrapped perfectly intact.
An object of this invention is to provide a method for the immobilization of an enzyme, which method permits the enzyme to be stably entrapped within a matrix of a photosensitive resin while minimizing the possibility of depriving the enzyme of its activity, wherein the enzyme consequently immobilized in the matrix without impairment of its enzymatic function is allowed to react effectively upon a substrate.